Identification of novel analgesic targets in ascending spinal projection neurons

项目概要/摘要 虽然慢性疼痛代表了一个巨大的公共卫生问题， 仅在美国，每年就有5600 - 6350亿美元，驱动神经元过度兴奋的分子机制 在伤害感受通路中的作用仍然不完全清楚。已经取得了重大进展， 阐明初级感觉神经元的遗传异质性及其在神经损伤后的可塑性 或组织损伤。然而，对这些神经元的全面分子概况知之甚少 尽管它们对疼痛有着明显的重要性， perception.更好地了解脊髓投射神经元内基因表达的完整模式 可以揭示新的循证策略，选择性地抑制脊髓伤害性网络的输出， 作为缓解慢性疼痛的一种手段。长期目标是更好地了解神经和组织损伤 改变中枢神经系统中伤害感受回路的功能。本申请的目的是识别损伤诱发的 脊髓投射神经元内基因表达的变化增强了它们在慢性疼痛下的放电 条件中心假设是，脊髓投射神经元的上行表现出独特的分子 表型，通过外周损伤显著调节以促进膜过度兴奋。的 所提出的工作的基本原理是，鉴定优先在脊髓中表达的基因， 投射神经元将产生新的药理学方法来抑制伤害性感受神经元的上行流动。 信息到大脑，同时最大限度地减少不必要的中断，全球感觉运动处理内 脊髓中心假设将通过追求以下具体目标来检验：（1）识别基因 在成年脊髓内的上行投射神经元中富集;和（2）阐明 慢性疼痛条件下投射神经元的基因表达增加膜兴奋性。 这些目标将通过使用翻译核糖体亲和纯化（TRAP）和下一代 RNA测序技术结合生物信息学、电生理学、免疫组织化学 和原位杂交方法。这项拟议中的工作是创新的，因为它将首次揭示， 将脊髓伤害感受回路连接到小鼠大脑的那些神经元的遗传表型， 关键参与神经性疼痛和炎症性疼痛的产生，以及阐明 该群体的分子特征在慢性疼痛状态期间改变。成果的 研究将是发现新的，脊髓投射神经元的细胞类型特异性标记物， 确定外周损伤可以放大伤害性感受“增益”的潜在遗传机制 在脊髓中传播。因此，该研究具有重要意义，因为它将揭示新的 这些分子靶点可以被操纵来选择性地沉默脊髓投射神经元， 损伤，以引起安全和有效的镇痛，同时最大限度地减少不良副作用。

Project Summary/Abstract While chronic pain represents a massive public health problem with a staggering economic cost of $560-$635 billion each year in the U.S. alone, the molecular mechanisms driving neuronal hyperexcitability within nociceptive pathways remain incompletely understood. Significant progress has been made towards elucidating the genetic heterogeneity of primary sensory neurons and their plasticity in the aftermath of nerve or tissue damage. However, much less is known about the comprehensive molecular profile of those neurons that convey nociceptive information from the spinal cord to the brain, despite their clear importance for pain perception. A better understanding of the complete pattern of gene expression within spinal projection neurons could reveal new evidence-based strategies to selectively dampen the output of the spinal nociceptive network as a means to alleviate chronic pain. The long-term goal is to better understand how nerve and tissue damage alter the function of nociceptive circuits in the CNS. The objective of this application is to identify injury-evoked changes in gene expression within spinal projection neurons that enhance their firing under chronic pain conditions. The central hypothesis is that ascending spinal projection neurons exhibit a unique molecular phenotype that is significantly modulated by peripheral injury to promote membrane hyperexcitability. The rationale for the proposed work is that the identification of genes that are preferentially expressed in spinal projection neurons will yield new pharmacological approaches to suppress the ascending flow of nociceptive information to the brain, while minimizing unwanted disruptions to global sensorimotor processing within the spinal cord. The central hypothesis will be tested by pursuing the following specific aims: (1) Identify genes that are enriched in ascending projection neurons within the adult spinal cord; and (2) Elucidate changes in gene expression in projection neurons under chronic pain conditions that increase membrane excitability. These aims will be accomplished by using translating ribosome affinity purification (TRAP) and next generation RNA sequencing techniques in combination with bioinformatics, electrophysiological, immunohistochemical and in situ hybridization approaches. The proposed work is innovative because it will reveal, for the first time, the genetic phenotype of those neurons connecting the spinal nociceptive circuit to the mouse brain that are critically involved in the generation of neuropathic and inflammatory pain, as well as elucidate how the molecular signature of this population changes during the chronic pain state. The outcome of these investigations will be the discovery of new, cell type-specific markers of spinal projection neurons and the identification of potential genetic mechanisms by which peripheral injuries can amplify the “gain” of nociceptive transmission in the spinal cord. As a result, the proposed research is significant because it will reveal novel molecular targets which could be manipulated to selectively silence ascending spinal projection neurons after injury, in order to evoke safe and effective analgesia while minimizing undesirable side effects.